Rho-ROCK Signal Pathway Regulates Microtubule-Based Process Formation of Cultured Podocytes – Inhibition of ROCK Promoted Process Elongation

Abstract

Background: Podocytes, renal glomerular visceral epithelial cells, have two kinds of processes, namely major processes containing microtubules (MTs) and foot processes with actin filaments (AFs). The present study investigated how MTs are organized by the Rho-ROCK signal transduction pathway during process formation of podocytes. Method: After induction of differentiation, podocytes of the conditionally immortalized mouse cell line were treated with Y-27632, a specific inhibitor of ROCK, and exoenzyme C3, an inhibitor of RhoA, as well as with forskolin whose effects include inhibition of RhoA, in order to inhibit the Rho-ROCK pathway. Results: Inhibition of ROCK significantly enhanced the formation of thick processes containing MT bundles. Y-27632 promoted process formation even in the presence of latrunculin A which disrupts AFs, strongly suggesting that ROCK directly regulates MT assembly. Treatment with Y-27632 increased MT stability, and stabilized MTs preferentially localized in podocyte processes. Moreover, when treated with a combination of Y-27632 and forskolin, and with Y-27632 and C3 as well, podocytes developed not only MT-based thick processes but also AF-based thin projections. Conclusions: These data indicate a contribution of ROCK in MT organization to promote podocyte process formation, although it was originally thought to regulate AF assembly. AF-based thin projections seem to be induced mainly by inhibition of RhoA and ROCK. The present study reveals a significant role of the Rho-ROCK signal pathway in the reorganization of both MTs and AFs during process formation of podocytes.

The microtubule end-binding protein EB2 is a central regulator of microtubule reorganisation in apico-basal epithelial differentiation

“…S3A and data not shown). Furthermore, inactivation of the Rho effector ROCK with Y27632 in ARPE-19 cells, which is noted for inducing microtubule stability, resulted in microtubule bundling and formation of microtubule-rich cell protrusions as previously reported (Darenfed et al, 2007;Gao et al, 2004) but also led to a significant increase in EB1 lattice association compared to untreated controls (supplementary material Fig. S3B).…”

“…Microtubule depolymerisation by Nocodazole, for example, activates RhoA/ROCK and destabilises microtubules due to release of the Rho effector GEF-H1 from microtubules. RhoA/ ROCK inhibition, on the other hand, induces microtubule stability and bundle formation as well as reducing stress fibres (Chang et al, 2008;Cheng et al, 2012;Gao et al, 2004;Kadir et al, 2011;Krendel et al, 2002;Scaife et al, 2003;Takesono et al, 2010).…”

Rho-kinase inhibition prevents proteinuria in immune-complex-mediated antipodocyte nephritis

“…Activation of Rho-kinases and cytoskeletal rearrangement (stress fiber formation) had been reported previously in cultured podocyte under sheer stress (13). Rho-kinase inhibition prevented these changes and was also shown to enhance process formation in culture (14).…”

“…Several specific Rho-kinase effectors such as ezrin/radixin/moesin (ERM), adducin, LIMK-1, or the regulatory subunit of the myosin-specific phosphatase have been identified (2). Upon activation, these effectors contribute to stress fiber formation and cell body contraction by various mechanisms (1,14,22). Many of the known effects of Rhokinases on cytoskeletal rearrangement have been found by Rho-kinase inhibitor experiments.…”

“…In podocytes, studies by Gao et al (14) and Kobayashi et al (22) demonstrated a significant role of the Rho/Rho-kinase signaling pathway in the reorganization of both microtubules and actin filaments during process formation in vitro. In their studies, Rho/Rho-kinase inhibition enhanced the formation of thick processes containing microtubular bundles and actin filament based thin projections, suggesting that downregulation of Rho-kinase activation allowed microtubule and actin assembly.…”